Changes in the Subchondral Bone, Visfatin, and Cartilage Biomarkers after Pharmacological Treatment of Experimental Osteoarthritis with Metformin and Alendronate.

Subchondral bone that has intense communication with the articular cartilage might be a potential target for pharmacological treatment in the early stages of osteoarthritis (OA). Considering the emerging data about the role of adipokines in the pathogenesis of OA, the administration of drugs that influence their level is also intriguing. Metformin and alendronate were administered in mice with collagenase-induced OA (CIOA) as a monotherapy and in combination. Safranin O staining was used for the assessment of changes in subchondral bone and articular cartilage. Before and after treatment, serum levels of visfatin and biomarkers of cartilage turnover (CTX-II, MMP-13, and COMP) were assessed. In the current study, the combined administration of alendronate and metformin in mice with CIOA led to the protection against cartilage and subchondral bone damage. In mice with CIOA, metformin led to a decrease in visfatin level. In addition, treatment with metformin, alendronate, or their combination lowered the level of cartilage biomarkers (CTX-II and COMP), while the level of MMP-13 was not influenced. In conclusion, personalized combination treatment in OA according to clinical phenotype, especially in the early stages of the disease, might lead to the identification of a successful disease-modifying therapeutic protocol in OA.

Assessment of the exposure of two pesticides on common carp (Cyprinus carpio Linnaeus, 1758): Are the prolonged biomarker responses adaptive or destructive?

Chlorpyrifos (CPF) and cypermethrin (CYP) are two insecticides that have a proven negative effect on non-target aquatic organisms when they enter the surface waters. However, literature on the comparative effects of these pesticides on important aquaculture fish species, such as common carp (Cyprinus carpio Linnaeus, 1758) is not yet scientifically detailed, especially over the long-term. The idea of conducting a long-term exposure is to find out how the observed biomarkers would change compared to the short-term exposure. In the natural environment, toxicants are not present alone, but in combination. By monitoring the long-term impact of individual substances, the state of aquatic ecosystems exposed to various toxicants could be predicted. Thus, this study aimed to evaluate the toxicity of different concentrations of CYP (0.0002, 0.0003, and 0.0006 µg/L) and CPF (0.03, 0.05, and 0.10 µg/L) in 50-L glass tanks on C. carpio, exposed for 30 days under laboratory conditions. A set of histological and biochemical biomarkers in the gills and liver were applied with the chemical analyses of water and fish organs. Furthermore, the condition and hepatosomatic index were calculated to assess the physiological status of the treated carps. The behavioral responses were also monitored, and the respiration rate was analyzed. The results suggest that CYP had a more prominent effect on the histological structure of fish organs, biochemical responses of anti-oxidant enzymes, behavior, and respiration rate compared to the effect of CPF. In addition, the results also indicate that the liver is more susceptible to chronic and chemically induced cellular stress compared to the gills, with overall destructive changes in the histological biomarkers rather than adaptive. Regardless of the scenario, our results provide novel insights into pesticide exposure and the possible biological impacts on economically important freshwater fish, exposed to lower CYP and CPF concentrations, based on the EU legislation (maximum allowable concentrations, MAC-EQS).

Biomarkers for pollution in caged mussels from three reservoirs in Bulgaria: A pilot study.

The mussel-watch concept was firstly proposed in 1975, which was later adopted by several international monitoring programs worldwide. However, for the very first time, a field experiment with caged mussels was performed in three reservoirs in Bulgaria to follow the harmful effects of sub-chronic pollution (30 days) of metals, trace, and macro-elements, as well as some organic toxicants, such as polybrominated diphenyl ethers and chlorinated paraffins. Therefore, we studied the biometric indices, histochemical lesions in the gills, biochemical changes in the digestive glands (antioxidant defense enzymes, such as catalase, glutathione reductase, and glutathione peroxidase; metabolic enzymes, such as lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase, and the neurotransmitter cholinesterase), in addition to the DNA damage in the Chinese pond mussel, Sinanodonta woodiana (Lea, 1834) in Kardzhali, Studen Kladenets and Zhrebchevo reservoirs in Bulgaria. Significant correlation trends between the pollution levels, which we reported before, and the biomarker responses were established in the current paper. Overall, we found that both tested organs were susceptible to pollution-induced oxidative stress. The different alterations in the selected biomarkers in the caged mussels compared to the reference group were linked to the different kinds and levels of water pollution in the reservoirs, and also to the simultaneously conducted bioaccumulation studies.

Disease-Modifying Potential of Metformin and Alendronate in an Experimental Mouse Model of Osteoarthritis.

Osteoarthritis (OA) is the most common degenerative joint disease causing progressive damages of the cartilage and subchondral bone, synovial inflammation, and severe pain. Despite the complex pathomorphological changes that occur in OA, the approach to different forms of OA is standardized. The global results from pharmacological treatment are not satisfactory. Hence, this study aimed to explore the effects of metformin, alendronate, and their combination on OA development and progression in mice with collagenase-induced osteoarthritis (CIOA). Female ICR (CD-2) mice were randomized to five groups: control group, CIOA untreated, CIOA + metformin, CIOA + alendronate, and CIOA + metformin + alendronate. OA was induced by the intra-articular (i.a.) injection of collagenase. OA phenotype was analyzed by flow cytometry (bone marrow cell differentiation), ELISA (serum levels of the adipokines leptin and resistin), and histology (pathological changes of the knee joint). Treatment with metformin, alendronate, or their combination inhibited the expression of RANK and RANKL on osteoblasts and osteoclasts obtained by ex vivo cultivation of bone marrow cells in mineralization or osteoclastogenic media. In addition, metformin treatment was effective for the attenuation of fibroblast differentiation, but not of mesenchymal stem cells (MSCs), while alendronate had an opposite effect. The combination of metformin and alendronate had a suppressive effect on both MSCs and fibroblasts differentiation. Treatment with metformin, alendronate, and their combination decreased serum concentrations of leptin and resistin in the chronic phase of arthritis. The histopathological examination showed that compared with the untreated CIOA group (OA score 9), the groups treated with metformin (OA score 4) or alendronate (OA score 6) had lower scores for cartilage changes. Metformin combined with alendronate significantly decreased the degree of cartilage degeneration (OA score 2), suggesting that this combination might be a useful approach for the treatment of OA patients.

Serum Leptin and Resistin Levels in Knee Osteoarthritis-Clinical and Radiologic Links: Towards Precise Definition of Metabolic Type Knee Osteoarthritis.

Obesity is considered a major risk factor for the development and progression of knee osteoarthritis (OA). Apart from the mechanical effect of obesity via increase in mechanical overload of weight-bearing joints, an association with hand OA has been observed. There has been increasing interest in the role of adipokines in the pathogenesis of OA in the recent years. It has been suggested that their systemic effects link obesity and OA. In this regard, the aim of the current study was measurement and analysis of serum levels of leptin and resistin in patients with knee OA with different body mass index (BMI). Seventy-three patients with primary symptomatic knee OA at the age between 35 and 87 years (mean age 66 years) were included in the study (67 women and 6 men). The patients were from 2nd to 4th radiographic stage according to Kellgren-Lawrence scale. 43 patients were with concomitant obesity (BMI ≥ 30 kg/m2, mean values 38.34 ± 8.20) and 30 patients with BMI < 30 kg/m2 (mean values 25.07 ± 2.95). Eleven individuals with different BMIs, including cases with obesity but without radiographic knee OA, were examined as a control group. Serum levels of leptin and resistin were measured via ELISA method. In patients with knee OA and BMI ≥ 30 kg/m2, serum levels of leptin (39.546 ± 12.918 ng/mL) were significantly higher as compared with healthy individuals (15.832 ± 16.531 ng/mL, p < 0.05) and the patients with low BMI (p < 0.05). In patients with BMI < 30 kg/m2 the levels of leptin (13.010 ± 10.94 ng/mL) did not differ significantly from the respective values in the control group (p = 0.48). Serum levels of resistin were also higher in knee OA patients in comparison with healthy controls, but the difference was statistically significant only for patients with high BMI (2.452 ± 1.002 ng/mL in the group with BMI ≥ 30 kg/m2; 2.401 ± 1.441 ng/mL in patients with BMI < 30 kg/m2; 1.610 ± 1.001 ng/mL in the control group, p < 0.05). A correlation was found between the serum levels of leptin and radiographic stage of OA, i.e., higher leptin levels were present in the more advanced 3rd and 4th radiographic stage, while for resistin a correlation was observed in the patient subgroup with BMI < 30 kg/m2. Serum leptin and resistin levels and clinical characteristics were analyzed in patients with different clinical forms of OA. Novel clinical correlations have been found in the current study in patients with isolated knee OA vs. cases with presence of other disease localizations. It has been observed that patients with isolated knee OA were significantly younger and had higher BMI as compared with cases in whom OA is combined with other localizations i.e., spondyloarthritis ± presence of hip OA and with generalized OA. This supports the hypothesis that presence of obesity promotes earlier development of knee OA as an isolated localization of the disease in younger patients before appearance of osteoarthritic changes at other sites. The levels of leptin and resistin in isolated knee OA were also higher. Serum levels of leptin and resistin in combination with patients' clinical characteristics suggest existence of different clinical and laboratory profile through which more precise definition of metabolic phenotype of knee OA would be possible. Considering the fact that obesity is a modifiable risk factor that has an impact on progression of knee OA, different approaches to influence obesity may offer potential for future disease-modifying therapeutic interventions.

Which Is More Toxic? Evaluation of the Short-Term Toxic Effects of Chlorpyrifos and Cypermethrin on Selected Biomarkers in Common Carp (Cyprinus carpio, Linnaeus 1758).

The general aim of this study was to investigate the negative short-term effects of different concentrations of chlorpyrifos (CPF) and cypermethrin (CYP), based on the EU legislation (MAC-EQS) in common carp (Cyprinus carpio Linnaeus, 1758) under laboratory conditions and to compare their toxicity. The fish were exposed to the pesticides for 96 h and then different histological and biochemical biomarkers were investigated in the gills and liver, and bioaccumulation analyses were conducted. The chemical studies showed increased pesticide concentrations in the gills as the first site for pollutants compared to the liver at the 96th hour. In addition, the histological analyses showed severe alterations in the gills and liver after exposure to both tested pesticides. In the gills, we found mainly intense proliferative and, to a lesser extent, degenerative changes and alterations in the circulatory system, such as necrosis and vasodilation. In the liver, regressive and progressive lesions, as well as circulatory disturbances and inflammation, were observed. The regressive lesions showed a higher degree of expression compared to the other changes. Furthermore, we found altered enzymatic activities-catalase, glutathione reductase, and glutathione peroxidase-in the liver, compared to the control. Overall, both tested pesticides impacted the studied biomarkers in common carp, even at concentrations lower than those permitted by law. However, the results of the comparative analysis showed a relatively higher toxicity of CYP compared to CPF in the fish. Still, questions persist as to whether the observed changes are adaptive or entirely destructive. To avoid any danger or risk, these pesticides must be applied cautiously, especially near water bodies.

Histological Alterations in Common Carp (Cyprinus carpio Linnaeus, 1758) Gills as Potential Biomarkers for Fungicide Contamination

ABSTRACTThe present study aimed to investigate the histological alterations in common carp gills caused by a fosetyl-Al and fenamidone based fungicide tested in laboratory conditions at 30, 38 and 50 mg/L concentration. In general, all the tested concentrations activated compensatory-adaptive mechanisms, which caused pathological changes in the fish gills. Results showed different histological alterations in the gill structure, which included lamellar lifting, edema, proliferation of the glandular cells and epithelium, covering the gill filament, fusion and degenerative alterations. Blood circulatory system showed vasodilatation of the secondary lamellae and aneurysms. Overall, there was enhancement of the gill histological changes, which was dose-dependent, i.e., proportional to the increasing fungicide concentrations. Thus, based on the results, it was concluded that the histological alterations in common carp gills could be applied as possible biomarkers in risk assessment and monitoring programs for pesticide contamination of aquatic ecosystems.

Histopathological Alterations in Common Carp (Cyprinus carpio L.) Gills Caused by Thiamethoxam

This work aimed to study the effect of new neonicotinoid thiamethoxam on the histological structure of common carp (Cyprinus carpio, L.) gills. Three experimental groups of fish were treated with 6.6, 10 and 20 mg/l thiamethoxam in laboratory conditions. Results showed different histological changes in the gill epithelium, which included lamellar lifting, edema, proliferation of the glandular cells and epithelium, covering the gill filament, fusion and degenerative alterations. The blood circulatory system showed mainly vasodilatation. All thiamethoxam concentrations activated compensatory-adaptive mechanisms, which caused pathological changes in the gills. Moreover, there was a tendency towards the enhancement of the gill histological changes, whose degree of expression was proportional to the increasing thiamethoxam concentrations.

Metal bioaccumulation in common carp and rudd from the Topolnitsa reservoir, Bulgaria.

Concentrations of arsenic (As), cadmium (Cd), copper (Cu), nickel (Ni), lead (Pb), and zinc (Zn) were determined in water samples and five fish organs (gills, liver, kidney, spleen, and muscle) of common carp (Cyprinus carpio L.) and common rudd (Scardinius erythrophthalmus L.) from the Topolnitsa reservoir (Bulgaria) in three seasons (spring, summer, and autumn). This water ecosystem is located in a copper mining and metallurgical region. Water metal concentrations were significantly higher in the summer than in the spring (p<0.05). Moreover, As, Cd, Cu, and Zn concentrations were higher than the national limits. Qualitative factors "element" and "fish organ" had a stronger influence on metal bioaccumulation than the factors "season" and "fish species". In fish, the highest metal levels were detected in the liver, spleen, kidney and gills, and the lowest in the dorsal muscle. Tissue levels were higher in the summer, but in general they were similar between the two Cyprinid fish. Fish muscles had the lowest metal levels at all times, but As and Pb exceeded the national and international standards. Therefore, we would not recommend fish consumption from Topolnitsa, as continuous metal contamination of the reservoir may seem to present human health risk.

Expression of leptin, NGF and adiponectin in metabolic syndrome.

Adipose tissue secretes a variety of adipokines involved in the regulation of energy metabolism and insulin resistance. Metabolic syndrome corresponds to a clinical condition in which white adipose tissue is characterized by an increased production and secretion of inflammatory molecules which may have local effects on adipose tissue physiology but also systemic effects on other organs. The aim of this study was to assess the expression of leptin, NGF and adiponectin in women with metabolic syndrome compared to healthy controls. Plasma leptin, NGF and adiponectin levels were measured by the ELISA method. Leptin and NGF immunohistochemical expression was analyzed in subcutaneous adipose tissue. The results indicated that in women with metabolic syndrome waist circumference, body mass index, HOMA index, glucose, total cholesterol and triglyceride levels were significantly increased in parallel with overxpressed plasma levels of leptin and NGF and decreased adiponectin. The immunohistochemical expression of leptin and NGF was very strong. In conclusion, this is the first study demonstrating a complex of immunochemical and immunohistochemical expression of the key adipokines including leptin, NGF and adiponectin in women with metabolic syndrome. Locally-produced pro-inflammatory adipokines probably contribute to the ethiopathogenic mechanisms ofmetabolic syndrome.

Morphological characteristics of neonatal adipose tissue.

The aim of the present work was to study the morphological characteristics of neonatal adipose tissue using rats as an animal model. The results revealed that the subcutaneous adipose tissue of newborns consists of packets of unilocular adipose cells (one large lipid drop occupying the whole cell and pushing the cytoplasm and the nucleus to the cell periphery) and some multilocular fat cells (several lipid droplets of different size and an almost centrally located nucleus). All the adipocytes demonstrated positive immunohistochemical expression for leptin, whereas the multilocular adipose cells were positive for cyclin D1. These findings suggest that the multilocular adipose cells are preadipocytes that have not yet finished proliferation and differentiation and could under some external and/or internal stimuli conclude their development and become mature unilocular adipocytes, thus increasing fat mass.